2 Materials and Methods
2.2 Methods
2.2.2 Protein manipulation methods
2.2.2.1 Protein isolation from eukaryotic cells
For protein isolation, eukaryotic cells were harvested from cell culture vessel using an appropriate protocol (chapter 2.2.4.2). Nuclear and cytoplasmic protein extracts were isolated using the NE-PER® Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Scientific) according to manufacturer‟s instructions.
2.2.2.2 Protein isolation from yeast cells
For protein isolation from yeast cells, 8 ml of SD medium with addition of adenine and histidine (chapter 2.1.10.1) was inoculated with a single yeast colony from a −LT agar plate and incubated (16–18 h) at 30°C by shaking at 250 rpm overnight. The overnight culture (OD600 ~1.0) was centrifuged at 1000 x g for 5 min at RT. The yeast cell pellet was resuspended in 500 µl of 2 M chilled LiAc and incubated for 5 min on ice. After centrifugation at 1000 x g for 5 min at RT, the cell pellet was resuspended in 100 µl of 0.4 M chilled NaOH, incubated for 5 min on ice and centrifuged at 1000 x g for 5 min. The cell pellet was resuspended in 100 µl of sample buffer containing 25 µl of NuPAGE® LDS Sample Buffer (4x) (Invitrogen), 10 µl of 1 M DTT and 65 µl of Ampuwa. After yeast proteins were denatured for 10 min at 70°C, the mixture was placed for 5 min on ice and was further centrifuged at 16,000 x g for 1 min to pellet cell debris. The protein containing supernatant was transferred into a new reaction tube and 28 µl of the supernatant was used for SDS-PAGE (chapter 2.2.2.6).
2.2.2.3 Protein concentration from cell culture medium
Proteins from cell culture medium were concentrated using the Vivaspin® Turbo 15 centrifugal concentrators (30 kDa MWCO) (Sartorius) according to manufacturer‟s instructions.
2.2.2.4 Determination of protein concentration
Protein concentration was determined according to the method of Bradford (1976). The used solution Roti®-Nanoquant (5x) (Carl Roth) consists of the Coomassie Brilliant Blue dye that has the property to bind unspecifically to proteins in acidic solution, resulting in a shift of its absorption maximum from 465 nm to 595 nm.
A Bovine Serum Albumin Standard Ampule (Thermo Scientific) was diluted to generate standard protein concentrations for a calibration curve to determine the protein concentration by extrapolating to this curve.
To determine protein concentration 5x Roti-Nanoquant was diluted with ddH2O to a final 1x concentration and further stored at 4°C up to 3 month. Protein sample was diluted 1:100 with Ampuwa and 50 µl of the sample was pipetted into a 96-well plate in triplicate.
Ampuwa was used as control. Each diluted protein sample was mixed with 200 µl of 1x Roti-Nanoquant and incubated for 5 min at RT. The absorptions were measured using theSynergyTMMxplatereader(BioTek)and calculated with the provided Gen5TM software.
2.2.2.5 Co-immunoprecipitation
Co-immunoprecipitation (Co-IP) is a technique to identify protein-protein interactions by using a target protein antibody for the indirect capture of proteins that are bound to the target protein. These protein complexes can be further analyzed for example by western blot analysis to identify unknown complex members.
To pre-clear isolated protein extract of proteins which can bind unspecifically to the beads, 500–1000 µg of protein extract and 20 µl of Protein G Magnetic Beads (New England Biolabs) were gently mixed, filled up with lysis buffer (chapter 2.1.6) to a total volume of 500 µl and rotated on the Stuart® SB2 rotator (Bibby Scientific) for 1 h at 4°C. Afterwards, a magnetic field was applied for 1 min by placing the reaction tube into a 6-Tube Magnetic
Separation Rack (New England Biolabs) to pull the beads to the side of the tube. The supernatant was pipetted into a new reaction tube, 2 µg of appropriate antibody (chapter 2.1.11.1) was added and the mixture was incubated on the rotator at 4°C overnight.
No antibody was added to the negative control. The following day, 20 µl of Protein G Magnetic Beads was added and the mixture was incubated on the rotator for 2 h at 4°C.
After brief centrifugation, a magnetic field was applied for 1 min and the supernatant was removed. The beads were washed with 500 µl of lysis buffer and incubated on the rotator for 5 min at 4°C. The mixture was briefly centrifuged and the washing step was additionally repeated 4 times as previously described. For the final washing step, 500 µl of TBS buffer was used. Thereafter, a magnetic field was applied for 1 min, the supernatant was removed and the bead pellet was gently mixed with 16.3 µl of lysis buffer, 6.25 µl of LDS Sample Buffer (4x) and 2.5 µl of 1 M DTT. Proteins were denatured for 10 min at 70°C and separated from the beads meanwhile. The mixture was placed for 5 min on ice and after applying a magnetic field for 1 min, the supernatant was completely used for SDS-PAGE (chapter 2.2.2.6).
2.2.2.6 SDS polyacrylamide gel electrophoresis
SDS polyacrylamide gel electrophoresis (SDS-PAGE) was used for the separation of proteins according to their molecular weight based on the method described by (Laemmli, 1970). The method bases on the use of gels and buffers with an operating pH of 7.0. This neutral pH increases the stability of both proteins and gels and leads to a better result of electrophoresis.
In general, samples consisted of 30 µg protein, 0.25 volume LDS Sample Buffer (4x) and 10% 1 M DTT (reducing agent), filled up with Ampuwa to a total volume of 20–28 µl, depending on the maximum sample loading volume of the gel. Proteins were denatured for 10 min at 70°C and placed on ice for 5 min. After brief centrifugation, protein samples were loaded onto a gradient gel (NuPAGE® 3–8% Tris-Acetate Gel or NuPAGE® 4–12%
Bis-Tris Gel, Invitrogen) placed in Novex® Mini-Cell Electrophoresis System (Invitrogen).
Pre-stained molecular weight standard (10 µl of SeeBlue® Plus2 Pre-Stained Protein Standard or 20 µl of HiMarkTM Pre-Stained High Molecular Weight Protein Standard, Invitrogen) was additionally loaded on the gel for determination the size of the separated proteins. Gel electrophoresis was performed for 10 min at 80 V and further at either 120 V (10-well gel) or 160 V (20-well gel) and depending on the composition of the gel, in
1x NuPAGE® Tris-Acetate SDS Running Buffer or 1x NuPAGE® MES SDS Running Buffer (Invitrogen).
2.2.2.7 Transfer of proteins from a polyacrylamide gel to a membrane
For detection of proteins by western blot analysis, separated proteins from a polyacrylamide gel were transferred to a membrane based on the method described by (Gershoni and Palade, 1983). Proteins <150 kDa were transferred on a nitrocellulose membrane (AmershamTM ProtranTM Supported 0.45 µm NC, GE Healthcare), whereas proteins >150 kDa were transferred on a PVDF membrane (AmershamTM HybondTM-P PVDF Transfer Membrane 0.45 µm, GE Healthcare).One membrane and four Blotting Paper Sheets (Sartorius) were cut to the size of the gradient gel. The nitrocellulose membrane was activated in ddH2O and further equilibrated in chilled transfer buffer I (chapter 2.1.6), while the PVDF membrane was activated in 100% methanol for approximately 30 s and then equilibrated in chilled transfer buffer II (chapter 2.1.6) for 5 min. Blotting papers and foam pads were soaked in appropriate transfer buffer. For transfer, the tank blotting system Mini Protean® II Cell (Bio-Rad) was used and the components were placed in a provided cassette on top of each other as follows: two sheets of blotting paper, membrane, polyacrylamide gel and two sheets of blotting paper. Potential air bubbles were carefully removed. The cassette was inserted into the tank in the way that the gel was located near to the cathode of the blotting system.
The tank further contained appropriate and chilled transfer buffer and a cooling unit. The transfer of proteins <150 kDa was performed at 250 mA per gel for 1 h at 4°C. The transfer of proteins >150 kDa was performed at 25 V overnight. Additionally, the buffer tank was put in an ice box and the buffer was agitated by a magnetic stirrer to avoid the formation of an ion gradient and to insure homogeneity of the buffer temperature.
2.2.2.8 Protein detection on membranes using antibodies
After protein transfer to a membrane, unspecific binding sites were blocked by incubating the membrane, depending on the used antibody, with blocking solution I or II (chapter 2.1.6) for at least 1 h at RT. Primary antibody (chapter 2.1.11.1) was diluted, depending on the antibody, in antibody diluent solution I or II (chapter 2.1.6) at 4°C overnight. The
following day, the membrane was washed with TBST buffer three times for 10 min to remove unbound antibody. The membrane was further incubated with appropriate secondary antibody (chapter 2.1.11.2) diluted, depending on the antibody, in antibody diluent solution I or II for 1.5–2 h at RT. To remove unbound antibody, the membrane was washed three times for 10 min with TBST buffer. The ImmobilonTM Western Chemiluminescent HRP Substrate (Merck Millipore) was used to visualize membrane bound proteins and was prepared according to manufacturer‟s instructions. The membrane was covered with the prepared solution, placed between a plastic wrap and incubated for 5 min at RT. The chemiluminescent signals were digitally detected using FluorChem® Q (Alpha Innotech) and evaluated with the provided AlphaView® Q software (Alpha Innotech).